Isolation and Characterization of Pigments from Microorganisms Isolated from Marine Soil

 

P. Senthamil Selvi^1*, Priya Iyer²

¹Research Scholar, Department of Biotechnology, Women`s Christian College, Chennai-600006, India

²Associate Professor, Department of Biotechnology, Women`s Christian College, Chennai-600006, India

 

ABSTRACT:

Bacterial pigments have many applications in current day to day life. The pigments produced by chromobacteria can be used for applications in dairy, pharmaceutical, food etc. In this study, 3 pigments were isolated i.e. Yellow from Xanthomonas sp., Pinkish Red from Rhodotorula sp., Orange from Sarcina sp. pigmented bacterial isolates obtained from the soil, were used for pigment extraction study. To isolate and characterize the Pigment producing bacteria from marine soil. We studied that the pigment producing bacteria and to identify the colour producing pigments. Soil samples from Pondicherry, Cuddalore, Chennai, and Andhra sea coast were collected and used for isolation of microbes producing pigments. Purification of extracted pigments done by column chromatography, whereas identification and characterization of purified pigment done by UV-Visible spectrophotometry and GC/MS analysis etc. The pigment isolated from bacteria was used for the antimicrobial activity, antioxidant, and anticancer and transformation studies. The bacterial extracts of carotenoid pigment extracted and used as natural colorants for food products and for dying of cloth.

 

 

 

INTRODUCTION:

Carotenoids are a class of compounds that have colouring power and have been widely used in food industry, leading its market to full development. Carotenoids occur widely in nature and, in general, all fruits and vegetables of colour are good sources of these compounds. Microorganisms are the most versatile tools in biotechnology to produce variety of molecules including enzymes, antibiotics, organic acids and pigments. Recent studies have shown that microorganisms are a promising source for natural colors.

 

 

 

 

 

 

The presence of pigments has been reported among the entire microbial world including Bacteria, Fungi, Yeast, algae and protozoa. Industrial production of natural food colorants by microbial fermentation has several advantages such as cheaper production, easier extraction, higher yields through strain improvement, no lack of raw materials and no seasonal variations. Pigments are compounds with characteristics of importance to many industries. In the food industry they are used as additives, colour intensifiers, antioxidants, etc. Pigments come in wide variety of colors, some of which are water soluble [1]. Microorganisms are the most powerful creatures in existence and determine the life and death on this planet.

 

MATERALS AND METHODS:

Sample Collection:

Collection of Soil samples were collected from different marine sources of India such as (Pondicherry, Cuddalore, and Chennai, Tuticorin, and Andhra sea coast). After collection of soil samples further study was done at the Department of Biotechnology Women`s Christian College, Chennai, India. The collected soil samples were stored at 4 °C for further studies.

 

Isolation of pigment producing bacteria from Soil samples:

(Pondicherry, Cuddalore, and Chennai, Tuticorin, and Andhra sea coast)

 

Identification of pigment producing bacteria:

Morphological identification:

The bacteria were subjected to Gram staining and Morphological identification.

 

Culture maintenance:

The isolated pure culture was maintained in Nutrient Agar slant for further experimental use.

 

Biochemical Identification:

The isolated bacteria were subjected to the following biochemical tests i.e. Indole test, methyl red and voges proskauer test, citrate test, oxidase test, catalase test, triple sugar iron agar test, urease test, carbohydrate fermentation test.

 

Test for carotenoids in bacteria [2]:

The bacterial cell isolates were grown in Luria Bertini broth was extracted.For the examination of carotenoid pigment using UV-Visible spectroscopy ranging from 450nm.

 

Thin layer chromatography [3]:

Silica gel TLC plates are cut as per need. The bacterial pigment extracts, the carotenoid yellow, orange pink red pigment, spots observed were marked and R_f value determined.

 

Isolation of carotenoid pigments by column: chromatography[4]:

The bacterial pigments were subjected to column chromatography.The fractions collected was evaporated and the thickly concentrated carotene fractions are used for TLC. Stationary phase silica gel (100-200µm). Chloroform: methanol 95: 5.

 

Antimicrobial activity of the bacterial pigments [5]:

The antimicrobial activity was checked by 2 methods (phosphomolybdenum method and H₂O₂ scavenging assay) given below.

 

Total antioxidant activity by phosphomolybdenum method [6]:

The antioxidant activity was determined.

 

Hydrogen peroxide (H₂O₂) scavenging assay [7]:

The ability of the extracts to scavenge hydrogen peroxide was determined and calculated

 

% scavenged (H₂O₂) =

(A of control – A of test / A of control) X 100

Reducing power assay [8]:

A spectrophotometric method as determined by Ferreira et al., 2007 was used for the measurement of reducing the power of the sample.

 

GC-MS analysis:

The Clarus 680 GC was used in the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250μm df) and the components were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260°C during the chromatographic run. The 1μL of extract sample injected into the instrument the oven temperature was as follows: 60°C (2 min); followed by 300°C at the rate of 10°C min^-1; and 300°C, where it was held for 6 min. The mass detector conditions were: transfer line temperature 240 °C; ion source temperature 240°C; and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. The fragments from 40 to 600 Da. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS NIST (2008) library.

 

Conformation test for carotenoids [9]:

The bacterial cell isolates were extracted. The appearances of blue colour confirm the presence of carotenoids.

 

Transformation studies [9]:

The plasmid from the organisms producing the pigment was used to transform E. coli DH5α.

 

Anticancer activity of the extracts - MTT assay [10]:

Cancer cell lines were purchased from Cancer Institute, Chennai. The cells were grown in a 96 well plate in Dulbecco’s Modified Eagle Medium, supplemented with 10% Foetal Bovine Serum and antibiotics (Penicillin-G). About 200μl of the cell suspension was seeded in each well and incubated at 37^°C 48 hours with 5% CO2 for the formation of confluent monolayer. The monolayer of cells in the plate was exposed to various concentrations of the bacterial carotenoid pigment were incubated for 24hours.The cyto toxicity was measured using MTT (5mg/ml). After incubation at 37°C in a CO₂ incubator for four hours, the medium was discarded and 200μl of DMSO was added to dissolve the formazan crystals. The absorbance was read in a micro plate reader at 570nm.

 

Applications [11]:

The bacterial carotenoid pigment (2ml) extract from the purified compound from column chromatography was mixed with alum potassium aluminium sulphate (6%).The cotton fabric and thread was kept immersed in the solution for about 5 minutes and kept for drying.

 

Washing performance:

Dried cotton fabrics were soaked in the detergent solution for 20 minutes and then washed using tap water tap water and dried for 30 minutes. The bacterial pigment purification carotenoid pigments were applied as food colourants.

 

RESULTS AND DISCUSSION:

Media was prepared and serially diluted sample was spread on to a plate containing in nutrient agar media and was incubated at 37°C for hours. The results of Gram staining and biochemicals were used for the identification of the organism. [Table 1]

 

Table 1:  gram staining results

Pigment	Bacteria name	Identifications
Yellow	Xanthomonas sp.	Gram –ve rods
Orange	Sarcina sp.	Gram – ve rods
Pink red	Rhodotorula sp.	Gram – ve rods

 

Extraction of carotenoid pigments:

Yellow:

Xanthomonas sp   the bacterial carotenoid yellow pigment was extracted with methanol and it has been confirmed with uv-visible spectrophotometry.

 

Orange:

Sarcina sp    Extraction of carotenoid pigments. The bacterial carotenoid orange pigment was extracted with methanol and it has been confirmed with UV-Visible spectrophotometry.

 

 

 

 

 

 

 

Pink red:

Rhodotorula sp    extraction of carotenoid pigments. The bacterial carotenoid pink red pigment ware extracted with methanol and it has been confirmed with uv-visible spectrophotometry.

 

Thin layer chromatography[12]:

The average Rf value of pigment obtained from bacterial carotenoid pigment was found to be 0.99.which is comparable with the standard pigment Rf value which was observed as 0.97. Davraj et al., (2009) Rf value of carotenoids were in the range of 0.99 to 0.97 which are matching with the standard so yellow, orange, pink red was carotenoids.

 

 

A (Yellow)               B (Orange )             C (Pink red)

Fig. 1: Extraction of carotenoid pigments

 

Column chromatography [13]:

From the column chromatography the compounds were separated based on the differences in partitioning between mobile and stationary phases. (Pavia D.L et.al., 1999).The pigments obtained were purified yellow, orange and pink red.

Table 2: biochemical analysis

Pigments	Bacteria name	Indole	MR	VP	TSI	Urease	Simons citrate	Sugarfermentation			Catalase
Yellow	Xanthomonas  sp.	+	+	_	_	_	+	+	+	+	+
Orange	Sarcina  sp.	_	+	_	_	_	+	+	+	+	+
Pinkish red	Rhodotorula  sp.	_	+	_	_	_	+	+	+	+	+

 

 

Fig. 2: Zone of inhibition against pigmented bacterial sp.

 

Fig. 3: Total antioxidant activity by Phosphomolybdenum method

 

Antimicrobial activity [14]:

The comparison of antimicrobial efficacy in terms of zone of inhibition of pigment against gram positive and gram negative organism E. coli, Staphylococcus sp., Salmonella sp., and Streptococcus sp. It is found that the pigments were found to exhibit maximum zone of inhibition, 13.5 mm against Staphylococcus and E. coli,12.5 mm was exhibited the carotenoid pigments.

 

 

Total antioxidant activity by phosphor-molybdenum method [15]:

In total antioxidant activity by phosphomolybdenum method have maximum antioxidant bacterial carotenoid pigment.

 

Hydrogen peroxide (H ₂O₂) scavenging assay [16]:

Total antioxidant activity by hydrogen peroxide scavenging assay for carotenoid pigment was found to be good.

 

 

Fig. 4: Total antioxidant activity by H ₂O₂ scavenging assay

 

Reducing power assay [17]:

Reducing power assays carried on the pigments was found to be positive.

 

 

Fig. 5: Reducing power assay

 

Conformation test for carotenoid pigment (Williamsonet al., 2014):

The nature of the extracted carotenoid pigment sample was tested. The appearance of blue colour on addition of sulphuric acid indicated the presence of carotenoids.

 

GC MS results [18]  

Fig. 6: Pink red pigment producing Rhodotorula sp.

 

    

Fig. 7: Yellow pigment producing Xanthomonas sp.

 

    

Fig. 8: Orange pigment producing Sarcina sp.

GC-MS Chromatogram of the methanolic extracted pigment showed different peaks the highest peak was observed and identified. The results of GC MS were as above.

 

Transformation [19]:

Several experiments were carried out transformants were obtained in these experiments. A similar set of experiments was carried out with carotenoids. In this case, transformation was observed. This probably resulted in more efficient transformation. The blue white colonies were observed indicating transformed colonies.

 

 

Fig. 9: Transformed colonies shown by Transformation

 

Application in cotton cloth [20]:

The isolated bacterial purified pigments was applied to dye cotton cloth. The dye was applied to cotton fixed in potassium aluminium sulphate (alum) solution and kept for drying. The fabric retained the respective yellow, pink red colour. These pigments can be utilized in the textile industries replacing synthetic dyes, hence being more eco friendly.

 

   

 

 

Fig. 10: Colorization done in cotton cloth by different pigments

Anticancer activity [21]:

Sharma et al., Tested compound of bacterial carotenoid pigments showed the weakest anticancer activity cancer cell line as detected by the MTT assay they showed the lowest IC50 (the highest anticancer activity) against lymphoma cells and the highest IC50 (the lowest anticancer activity) orange pigment (Paracoccus spp) these organisms show high activity against cancer cells and can be used for anticancer treatment. [2] States that anticancer compounds from marine microorganisms inhibit cell growth in various cells through bacterial pigments.

 

 

 

 

Fig. 11: MTT assay for Anticancer activity

 

CONCLUSIONS:

The organism are isolated and characterized by colony morphology Gram staining and Biochemical tests. Natural colour Pigment were extracted from bacteria. Extraction of pigments isolated by column chromatography and TLC. Pigments were characterized by UV Visible spectrophotometry, GC/MS analysis. Pigments were tested against 1.Antimicrobial activity 2. Antioxidant and anticancer activity. The isolated organisms were used for Transformation studies. The extracted bacterial pigment was used for dyeing cotton cloth, fabric thread and food samples. The Natural applied bacterial pigment extracted was used for dying cotton cloth and fabric thread.

ACKNOWLEDGEMENTS:

We express my deep and sincere gratitude to my guide Dr. Priya iyer for her precious help and support.

 

CONTRIBUTION OF AUTHORS:

P. Senthamil selvi and Priya iyer are the equally contributed in this research article.

 

REFERENCES:

1.     Tibor, Liquid Chromatography of natural pigments and synthetic dyes. J. Chromatographic Lib., 2007; 71: 1-591.

2.     Jiang, Y., Chen, F. and Hyde, K.D., Production potential of water-soluble Monascus red pigment by a newly isolated Penicillium spp. J. Agr. Techn., 2005; 1(1): 113-126.

3.     Dufosse Manimala MR, Murugesan R. In vitro antioxidant and antimicrobial activity of carotenoid    pigment extracted from Sporobolomyces sp. isolated from natural source. J Appl Nat Sci,   2007; 83(12): 649-53

4.     Balraj J Sasidharan, P., R. Raja, C. Karthik, R. Sharma and IndraArulselvi P. Isolation and Characterization of yellow pigment producing Exiguobacterium sp., J Biochem Tech. 2012 876(32): 632-635.

5.     Duffose, L., Pigments Microbial Encyclopedia Microbial. Production and Food Applications of the red pigments of Monascus ruber. J. Food Sci. Carotenoids as Food Colorants, Cereal Food World, 2009; 376(4): 198-203.

6.     Ruch Williamson, N. R., P. C. Fineran, S. R. Cristwood, S. R. Chawral, F. J. Leeper and Salmond, Anticancer and Innunosuppressive properties of bacterial prodigionines. Future Microbial. 2007; 456(87): 605-618.

7.     Williamson Ferreira Keneni, A. and Gupta Characterization of a Red bacterium Strain isolated From Root Nodule of a Faba Bean (Vicia faba. L.) for Growth and Pigment production, J. Advanced Laboratory Research in Biology. 2011; 321(67): 138-146. Zhang Mekhael, R., and Yousif, The Role of Red Pigment Produces by Serratia marcescens as Antibacterial and Plasmid Curing Agent. The 2^nd Kurdistam Conference on Biological Sciences. Bacterial pigments, Microbiologist. 2009; 234(76): 268-274.

8.     Pattnaik, P., U.Roy, U and Jain, Biocolors, new Generation Additives For Food, Indian Food Industry. 1997; 654(87): 21-32.

9.     Jantas Evangelin Ezhil Malar S, et al. Extraction and characterization of the pigment violacein from Chromobacterium violaceum and its antibacterial properties. J Sci Technol 2014; 123(54): 30-52.

10.  Shiva Krishna Pabba Nakamura Y, Asada C, Sawada T. Production of antibacterial violet pigment by psychrotropic bacterium RT102 strain. Biotechnol Bioprocess Eng 2003; 432(32): 37-40

11.   Davraj Balraj J, Pannerselvam K, Jayaraman A. Isolation of pigmented marine bacteria Exiguobacterium sp. from peninsular region of India and a study on biological activity of purified pigment. Int J Sci Technol Res 2014; 654(78): 375-385.

12.  Pavia Sudhakar T, Karpagam S, Premkumar J. Biosynthesis, antibacterial activity of pyocyanin pigment produced by Pseudomonas aeruginosa SU1. J Chem Pharm Res 2015; 321(71): 921-924.

13.  Dufossel Bhat SV, Khan SS, Amin T. Isolation and characterization of pigments producing bacteria from various foods for their possible use as biocolors, International Journal of Recent Scientific Research. 2013; 127(54): 1605-1609.

14.  Balraj Goswami G, Chaudhuri S, Dutta D. Effect of pH and temperature on pigment production from an isolated bacterium, Chemical Engineering Transactions. 2010; 543(42): 127-132.

15.  Ruch Raj DN, Dhanasekaran D, Thajuddin N, Panneerselvam A. Production of Prodigiosin from Serratia marcescens and its Cytotoxicity Activity. J Pharma Res. 2009; 284 (73):590-593.

16.  Ferreira Williamson NR, Fineran PC, Cristwood SR, Chawral SR, Leeper FJ, Salmond GPC. Anticancer and Immunosuppressive properties of bacterial prodigionines. Future Microbial. 2007; 283(289):  605-618.

17.  Edge, R., Mcgarvey, D.J. and Truscott, T.G. The carotenoids as antioxidants: A review. Journal of Photochemistry and Photobiology, 1997; 41(87): 189-200.

18.  Murugesan 2009 Latha, B., Jeevaratnam, Murali, H.S. and Manja, K.S.  Influence of growth factors on carotenoid Pigmentation of Rhodotorula glutinis DFR-PDY from natural source. Indian Journal of Biotechnology. 2009; 4 3:  53 -357.

19.  (Zhang et.al., 1988) Scolnik PA, Bartley GE 1995 Nucleotide sequence of a putative geranylgeranyl pyrophosphate synthase from Arabidopsis. Plant Physiology 1995; 104: 1469-1470.

20.  Jantas Khanna SK, Singh GB Toxicity of commonly used food colors: A review. J. Sci. Indian Res (1975); 34: 631-635.

21.  Dhanasekaran Andrighetti-Frohner CR, Antonio RV, Creczynski-Pasa TV, Barardi CRM, Simoes CMO Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum Memórias do Instituto Oswaldo Cruz  (2003) ; 98: 843–848

 

 

 

 

Accepted on 20.07.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4296-4302.